1. Field of the Invention
This invention relates to laboratory equipment used for performing sequential chemical reactions of which the polymerase chain reaction (PCR) is an example. In particular, this invention relates to thermal cyclers for such reactions, and to methods and apparatus for controlling the temperature in each of a multitude of reaction vessels in which rapid and accurate temperature changes are needed.
2. Description of the Prior Art
PCR is one of many examples of chemical processes that require precise temperature control of reaction mixtures with rapid and precise temperature changes between different stages of the process. PCR itself is a process for amplifying DNA, i.e., producing multiple copies of a DNA sequence from a single strand bearing the sequence. PCR is typically performed in instruments that provide reagent transfer, temperature control, and optical detection in a multitude of reaction vessels such as wells, tubes, or capillaries. The process includes a sequence of steps that are temperature-sensitive, different steps being performed at different temperatures and the sequence being repeated a multitude of times to obtain a quantity large enough for analysis and study from an extremely small starting quantity.
While PCR can be performed in any reaction vessel, multi-well reaction plates are the reaction vessels of choice. In many applications, PCR is performed in “real-time” and the reaction mixtures are repeatedly analyzed throughout the process, using the detection of light from fluorescently-tagged species in the reaction medium as a means of analysis. In other applications, DNA is withdrawn from the medium for separate amplification and analysis. Multiple-sample PCR processes in which the process is performed concurrently in a number of samples can be performed by placing each sample in one well of a multi-well plate or plate-like structure and simultaneously equilibrating all samples to a common thermal environment in each step of the process. The samples can also be exposed to two thermal environments simultaneously to produce a temperature gradient across each sample. An alternative to multi-well sample plates are individual plastic tubes held together by a tube rack or support or simply individually placed in a common block of high thermal conductivity known as a “thermal block” (described below) that controls the temperature.
In the typical PCR instrument, either a multi-well plate (usually one with 96 wells in an 8×12 array, but often ones with larger or smaller numbers of wells) with a sample in each well or a series of individual plastic tubes is placed in contact with the thermal block. The thermal block is heated and cooled either by a Peltier heating/cooling apparatus, which may be a single Peltier module or an array of modules, or by a closed-loop liquid heating/cooling system that circulates a heat transfer fluid through channels machined into the block. In either case, the heating and cooling of the thermal block are typically under the control of a computer with input from the operator. The thermal block makes intimate contact with the plate wells or the tubes to achieve maximal heat transfer. The reaction vessels, whether they be a plate or individual tubes, are usually plastic which itself is not a medium of high thermal conductivity. The plastic itself, plus the interface between the plastic and the metallic thermal block, produces thermal resistance which must be reduced or at least controlled to achieve efficient heat transfer between the thermal block and the reaction media. Reduction and control of the thermal resistance can be achieved by applying force to the vessels to press the vessels against the corresponding depressions in the thermal block. The force must be applied evenly to achieve uniform temperature control and minimal thermal resistance. The same force also serves to help seal the vessels during the thermal cycling and to maintain the seal during the pressure changes that result from the heating and cooling stages of the thermal cycling. The force must be adequate to serve all of these purposes, and the thermal cycler, which term is commonly used to denote the instrument in which the entire PCR process is performed, must also be able to accommodate reaction tubes or plates of different heights, and also to allow the operator to select the magnitude of the force to be applied. The optimal thermal cyclers are those that are automatically operated with safeguards against user error.